PROJECT SUMMARY Globally, respiratory syncytial virus (RSV) is the single most important pathogen causing lower respiratory tract disease in the first year of life, and for which there is no vaccine. Previous vaccine strategies resulted in severe RSV disease in infant vaccine recipients upon primary infection. Over the past 60 years subsequent vaccines have failed. We, therefore, developed a neonatal murine model of RSV to interrogate the respiratory mucosal immune response against RSV. Our overarching goal is to identify age-dependent immune regulation of mucosal adaptive immune cells that results in susceptibility to RSV and can be targeted to precisely design vaccines for infants. Tissue-resident memory T cells have recently been discovered to provide sterilizing protection against influenza by rapidly responding to infection. CD8 T cells are critical for RSV clearance in murine models and are associated with reduction of virus and reduced symptomatology in humans. Our research and that of other show that neonatal CD8 T cell responses are often reduced compared to adults and poorly differentiate into memory cells. How RSV-specific tissue-resident memory T cells (TRM) develop in infants and if they can be induced to provide protection is unknown. Prior vaccines have not targeted this newly discovered T cell subset and they have not been explored for vaccine design. Our central hypothesis to be tested is that neonatal RSV-specific T cells poorly differentiate into TRM resulting in poor protective immunity, but that enhancing innate immune activation can improve neonatal RSV-specific T cell localization to the respiratory mucosa and establish protective RSV-specific TRM. Three Specific Aims are proposed to test this hypothesis, with the goal of identifying novel immune targets for RSV vaccines designed for neonates. The proposed innovative experimental approaches are designed to: (1) identify the requirements of the neonatal innate immune response to provide antigen and/or T cell activation to establish RSV-specific TRM, (2) identify the subspecialized antigen presenting cells that induce RSV-specific TRM and how to enhance APC activation to retain RSV-specific memory CD8 T cells in the respiratory mucosa, (3) define the role of RSV-epitope specific TRM to protect against RSV infection. These studies will define the key components of vaccine design including antigen availability and the critical antigen presenting cells to target in order to develop RSV-specific TRM in neonates that protect upon challenge.